The invention relates to a method for producing magnetic nanoparticles that comprise metal oxide polymer composites.
There are already a number of technically established applications for magnetic composite particles having diameters that can be measured in nanometers. For instance, such particles can be employed in molecular biological applications for isolating, fixing, and cleaning cells, cell constituents, nucleic acids, enzymes, antibodies, proteins, and peptides, in cellular biology for phagocytosis experiments, in clinical chemistry as a component of diagnostic assays or therapeutic pharmaceuticals, in clinical diagnostics as contrasting agents, radionuclide or drug carriers, in biochemistry and technical chemistry as solid phases for examining molecular recognition phenomena and heterogeneous catalytic processes.
A number of polymer-coated metal oxide particles for biological applications in magnetic fields have been described since the mid-1980s. In particular, magnetizable nanoparticles smaller than 200 nm unlock new possibilities for transporting and separating cells, cell constituents, bioactive molecules, and radionuclides (US2003/0099954 MILTENYI; WO01/17662 ZBOROWSKI; WO02/43708 ALEXIOU), for marking in contrasting magnetic imaging and diagnosis methods (US2003/0092029A1 JOSEPHSON; WO01/74245 JOHANSSON; U.S. Pat. No. 5,427,767 KRESSE), and the mechanical (DE10020376A1 KOCH) and thermal influencing of living cells (U.S. Pat. No. 6,541,039 LESNIAK) and have therefore been continuously improved in terms of their application-related properties. Common to all of the applications is the fact that magnetizable metal oxides having a biocompatible polymer coating to form composite particles having sizes from 5 nm to 500 nm are bound to a colloidally stable suspension with an aqueous base. The coating material should either prevent interaction with biological materials, facilitate good tolerance with living cells, and influence the paths for metabolization in living organisms, or should enable selective bonding to the surface using targeted functionalization with biochemically active substances, or should release enclosed substances in a controlled manner. An energetic interaction with external magnetic fields is used by means of the magnetizable portions of the composite particles. In magnetic fields, such particles, depending on the magnetic properties, experience an alignment and they move corresponding to physical magnetic field gradients and react to temporal changes in the external magnetic field. A great number of methods were described for producing iron oxide crystallites as metal oxide particles, for instance by sintering at high temperatures with subsequent mechanical comminution, cluster formation under vacuum conditions, or wet chemical synthesis from solutions. The precipitation of iron oxides can occur under non-aqueous conditions (U.S. Pat. No. 4,677,027 PORATH) and subsequently be converted to aqueous conditions (U.S. Pat. No. 5,160,725 PILGRIM) or can occur exclusively in aqueous solutions (U.S. Pat. No. 4,329,241 MASSART). An aqueous formulation is used for biological applications because of toxicological considerations (U.S. Pat. No. 4,101,435 HASEGAWA). Wet chemical synthesis of the iron oxide crystallites can precede coating with polymer components (core-shell method) or can occur in the presence of the polymer (one-pot method). The core-shell method requires that stabilizers be added to the iron oxides, since the latter tend to form aggregates in aqueous suspension. Stabilizers can be amphiphilic substances (WO01/56546 BABINCOVA) or additional nanoparticles with an electrically charged surface (U.S. Pat. No. 4,280,918 HOMOLA). Surface-active substances as stabilizers can severely limit the options for chemical functionalization of the surface, however. Today in general magnetizable nanocomposite particles containing iron that are produced primarily using the one-pot method are accepted for medical applications due to their physical and chemical properties and pharmaceutical/galenic stability. The one-pot method uses the coating polymer directly during the formation of the iron oxides for stabilization during nucleation and growth of the crystallites from the solution. One of the most frequently employed coating materials is dextrane in a number of modifications. However, other polysaccharides such as arabinogalactane, starch, glycosaminoglycanes, or proteins have also been used (U.S. Pat. No. 6,576,221 KRESSE). Precipitation of iron(II) and iron(III) salts in the presence of dextrane (U.S. Pat. No. 4,452,773 MOLDAY) is probably the simplest method. This method is modified by using ultrasound and subsequent thermal treatment in a flow-through method (U.S. Pat. No. 4,827,945 GROMAN). The quality of the product can be further improved using magnetic classification (WO9007380 MILTENYI). Further encapsulation/coating, generally while using amphiphilic substances as stabilizers, can substantially modify the behavior of biological systems with regard to the composite particles (U.S. Pat. No. 5,545,395 TOURNIER, EP0272091 ELEY).
For producing highly disperse aqueous systems as injectable liquid, special methods of homogenization are used in addition to various stabilizers. Such methods are for instance rotor/stator homogenization and high-pressure homogenization. Particularly high mechanical energy input is attained using liquid jet or a liquid slot-nozzle high-pressure homogenizers (micro fluidizer technology), which is used in particular for producing liposomes (U.S. Pat. No. 5,635,206 GANTER) but also in other cases facilitates the production of injectable active substance formulations (U.S. Pat. No. 5,595,687 RAYNOLDS). The use of a high-pressure homogenizers for producing oxidic nanocomposite particles by means of controlled coalescence with subsequent drying in emulsions whose non-aqueous components contain an oxide component as sol is described in conjunction with the industrial production of catalyst materials (U.S. Pat. No. 5,304,364 COSTA) and electrographic toner particles, ceramic powder, felt materials, spray coatings, active substance carriers, or ion exchange resins (U.S. Pat. No. 5,580,692 LOFFTUS).
None of the described magnetic particle types in the range of less than 200 nm can generally be concentrated or fixed without complex separating methods (e.g. high gradient magnet separation).
On the other hand, there are already numerous magnetic particle applications in the life sciences that could be performed with much greater efficiency using separating steps on permanent magnets or that require high magnetic mobility of the particles for other reasons.